Invasive aspergillosis (IA) is one of the most serious causes of morbidity and mortality among immune compromised patients. Mortality may exceed 50% despite aggressive antifungal therapy. A major cause of treatment failure is the difficulty in obtaining an early diagnosis that would facilitate timely antifungal therapy. Early diagnosis can markedly improve survival. The goal of this project is development of an immunoassay for detection of Aspergillus antigens in blood or urine to facilitate early diagnosis of IA. The difficulty in development of immunoassays for diagnosis is a determination of which of the potentially hundreds or thousands of fungal antigens produced in vivo will show up in body fluids in concentrations sufficient for detection. A novel approach to target discovery, termed In vivo Microbial Antigen Discovery (InMAD), will be used to identify candidate protein antigens for immunoassay. The InMAD strategy is based on the hypothesis that serum or urine from mice infected with Aspergillus fumigatus will contain precisely those fungal proteins that would be targets for immunoassay. Serum or urine from A. fumigatus-infected BALB/c mice will be collected and filtered to remove whole cells/hyphae but leave behind soluble antigens generated during infection. These soluble antigens are potential targets for immunoassay. The filtered samples will be used to immunize nave BALB/c mice. Nave BALB/c mice will see the fungal antigens as foreign and will make antibodies. Serum will be collected from the immunized mice and used to identify antigens recognized by the antibodies by use of one and two dimensional immunoblots prepared from the fungus. The study will occur in two phases. The first R21 phase will focus on target discovery. Potential diagnostic targets will be identified using the InMAD approach and targets will be validated on the basis of secretion into serum or urine in animal models of IA and a determination of specificity for Aspergillus spp. The second R33 phase will be immunoassay development and evaluation. Polyclonal and monoclonal antibodies will be produced to target proteins; immunoassays will be constructed that target the most promising proteins; and immunoassays will be evaluated as a means for diagnosis of invasive aspergillosis. The ultimate product will be an immunoassay that identifies the presence of two or more distinct Aspergillus- specific antigens. Proof of concept for the InMAD approach to target discovery is already in hand for tularemia, melioidosis and relapsing fever where candidate proteins and polysaccharides that are shed into serum during infection have been identified.